This invention relates generally to the high resolution imaging of features of very small objects utilising penetrating radiation such as x-rays. The invention is especially suitable for carrying out x-ray phase contrast microscopic imaging, and may be usefully applied to the ultra high spatial resolution imaging of microscopic objects and features, including small biological systems such as viruses and cells and possibly including large biological molecules.
A known approach to microscopy utilising x-rays is projection x-ray microscopy, in which a focussed electron beam excites and thereby generates a spot x-ray source in a foil or other target. The object is placed in the divergent beam between the target and a photographic or other detection plate. There have more recently been a number of proposals for using the electron beam of an electron microscope to excite a point source for x-ray microscopy. Integration of an x-ray tomography device directly into an electron microscope was proposed by Sasov, at J. Microscopy 147, 169, 179 (1987). Prototype x-ray tomography attachments for scanning electron microscopes using charge coupled device (CCD) detectors have been proposed in Cazaux et al, J. Microsc. Electron. 14, 263 (1989), Cazaux et al, J. Phys. (Paris) IV C7, 2099 (1993) and Cheng et al X-ray Microscopy III, ed. A. Michette et al (Springer Berlin, 1992), page 184. Ferreira de Paiva et al (Rev. Sci. Instrum. 67(6), 2251 (June 1996)) have developed and studied the performance of a microtomography system based on the Cazaux and Cheng proposals. Their arrangement was an adaptation of a commercially available electron microprobe and was able to produce images at around 10 xcexcm resolution without requiring major alterations to the electron optical column. The authors concluded that a 1 xcexcm resolution in tomography was feasible for their device. All system components and methods of interpretation of image intensity data in these works were based on the mechanism of absorption contrast.
A review article by W. Nixon concerning x-ray microscopy may be found in xe2x80x9cX-rays: The First Hundred Yearsxe2x80x9d, ed. A Michette and S. Pfauntsch, (Wiley, 1996, ISBN 0.471-96502-2), at ps 43-60.
The present applicant""s international patent publication WO 95/05725 disclosed various configurations and conditions suitable for differential phase-contrast imaging using hard x-rays. Other disclosures are to be found in Soviet patent 1402871 and in U.S. Pat. No. 5,319,694. Practical methods for carrying out hard x-ray phase contrast imaging are disclosed in the present applicant""s co-pending international patent publication WO 96/31098 (PCT/AU96/00178). These methods preferably involve the use of microfocus x-ray sources, which could be polychromatic, and the use of appropriate distances between object and source and object and image plane. Various mathematical and numerical methods for extracting the phase change of the x-ray wavefield at the exit plane from the object are disclosed in that application and also in Wilkins et al xe2x80x9cPhase Contrast Imaging Using Polychromatic Hard X-raysxe2x80x9d Nature (London) 384, 335 (1996) and our co-pending international patent application PCT/AU97/00882. The examples given in these references primarily related to macroscopic objects and features, and to self contained conventional laboratory type x-ray sources well separated in space from the sample.
It is an object of the present invention, at least in a preferred application, to facilitate x-ray phase contrast imaging of microscopic objects and features.
The invention entails a realisation that the objective just mentioned can be met by a novel approach in the adaptation of electron microscopes to x-ray imaging or by the use of intense laser sources or x-ray synchrotron sources to produce a microfocus x-ray source.
In a first aspect of the invention, there is provided a sample cell for use in x-ray imaging, including structure defining a chamber for a sample, and, mounted to the structure, a body of a substance excitable by an appropriate incident beam to generate x-ray radiation, the cell being arranged so that, in use, at least a portion of the x-ray radiation traverses the chamber to irradiate the sample therein and thereafter exits the structure for detection.
In one embodiment, the cell is an integral self-contained unit adapted and dimensioned to be inserted in complementary holder means, e.g. the sample stage, of a scanning electron microscope or microprobe at a position where the electron beam of the microscope or microprobe is focussed on the body of excitable substance, and thereby provides the incident beam for exciting the substance to generate x-ray radiation.
In another embodiment, the substance is excitable by an incident focussed beam of electromagnetic radiation, e.g. a laser beam or synchrotron radiation beam, to generate x-ray radiation.
The cell is preferably an array of layers, of dimensions parallel to the plane of the layers in the range a micron or so to a few e.g. 10 millimeters. The cell is advantageously adapted for use in phase contrast imaging in that said layers through which the excited x-ray radiation passes are highly homogeneous and have very smooth surfaces for preserving high spatial coherence of the incident beam in the radiation that irradiates the sample, and thereby optimising useful contrast in the image. This is especially desirable for the exit surface from the layer of said excitable substance, and for subsequent layers in the sample cell.
The excitable substance is preferably a layer of the substance applied to the structure defining the cell but may also be free standing. This structure preferably includes a substrate and/or spacer layer, transparent generally to x-rays or to a selected x-ray energy band(s), separating the layer of excitable substance from the sample. Although largely transparent to the radiation energy band(s) of interest, the substrate and/or spatial layer may also be chosen such as to be strongly absorbing for energies outside this band(s) in order to enhance the chromatic coherence of the x-ray beam contributing to the image.
The said cell may be open, or may be arranged to be hermetically sealed, eg. to permit evacuation of the electron-microscope chamber after placement of the sample in the chamber. The chamber or cell may be adapted to be enclosed and if so the structure includes an x-ray transparent window by which the said x-ray radiation exits the structure for detection.
The layer of excitable substance is preferably of a thickness in the range 10 to 1000 nm, and the separation of this layer from the sample may be in the range 1 to 1000 xcexcm.
In this first aspect, the invention extends to an x-ray microscope or microprobe, eg. a scanning x-ray microscope or microprobe, having means to generate a focussed electron beam, and a sample cell, as described above in any one or more of the variations described, retained in holder means at a position where said electron beam is focussed on said body of excitable substance and thereby provides said incident beam for exciting said substance to generate x-ray radiation. Preferably, for very high resolution imaging, the means to generate a focussed electron beam includes a field emission tip electron source.
In a second aspect, the invention provides a method of deriving a magnified x-ray image of one or more internal boundaries or other features of a sample, comprising:
disposing the sample in a sample cell according to the first aspect of the invention and fitting the cell into holder means of an electron microscope or microprobe at a position where the electron beam of the microscope or microprobe is focussed on said body of excitable substance and thereby provides said incident beam for exciting said substance to generate x-ray radiation;
irradiating the excitable substance with an electron beam to cause the substance to generate x-ray radiation, at least a portion of which traverses the chamber to irradiate the sample, including the one or more internal boundaries or other features, and thereafter exits the cell structure; and
detecting and recording at least a portion of said radiation after it has irradiated the sample, to provide an image of the one or more internal boundaries or other features of the sample.
The x-ray imaging may be absorption-contrast or phase-contrast imaging or both. The invention is especially suited to performance of phase contrast imaging. The image(s)) may be energy filtered by the detector system or other means, or may be simultaneously collected as a set of images corresponding to a series of x-ray energy bands.
The x-ray radiation generated by the excitable substance is preferably in the medium to hard x-ray range, ie. in the range 1 keV to 1 MeV, and may be substantially monochromatic, or polychromatic. In the former case, the method may further include enhancing the degree of monochromaticity. In the practice of the method or use of the apparatus, the sample to image plane distance is preferably of the order of 10 to 200 mm.
In a still further aspect, the invention provides an x-ray microscopic imaging configuration comprising means to support a sample, a body of a substance excitable by an appropriate incident beam to generate x-ray radiation, said body being retained on a substrate disposed in use between said body and said sample and thereby serving as a spacer; and means to adjust the relative position of said sample and said body.